Antisense oligonucleotides have long held the potential to decrease the expression of a targeted gene by inhibiting transcription or translation and thereby achieve a phenotypic effect based upon the expression of that gene. However, many of the effects achieved by these oligonucleotides may not be mediated by inhibition of the target gene (C. Stein and Y.-C. Cheng, Science 261, 1004-1012 (1993); R. Wagner, Nature 372, 333-335 (1994)).
The erbB-2 gene codes for a 185kd tyrosine kinase linked transmembrane protein which is overexpressed in 30-50% of primary breast cancers (D. Slamon et al., Science. 244, 707-712 (1989); M. Press et al., Prog. Clin. Biol. Res. 354A, 209-221 (1990); M. Berger et al., Cancer. Res. 48, 1238-1243 (1988); D. Allred et al., Hum. Pathol 23, 974-979 (1992)). Overexpression, which is frequently due to gene amplification, is an early event in the development of many breast cancers and is maintained during invasion and metastatic progression of the disease (J. D. Iglehart et al., Cancer. Res. 50, 6701-6707 (1990)). Expression of erbB-2 is low in most normal adult tissues making it an attractive therapeutic target (M. Press et al., Oncogene. 5, 953-962 (1990)). We recently described a set of methods for delivering phosphorothioate oligonucleotides and measuring antisense activity against the human erbB-2 oncogene in breast cancer cells (J. Vaughn et al., Proc. Natl. Acad. Sci. U. S. A. 92, 8338-8342 (1995)). Antisense oligonucleotides or control sequences are co-delivered to cells with a fluorescent tagged oligonucleotide using cationic liposome mediated transfer. A high concentration of the fluorescent tracer and non-tagged oligonucleotide rapidly accumulate in the nucleus. Cells receiving the co-delivered oligonucleotides can then be identified, quantitated, immunostained for the level of the antisense target protein, and physically sorted to measure RNA levels and phenotypic changes. Because flow cytometric analysis is quantitated on a per cell basis, simultaneous two-color analysis of the tagged oligonucleotide (a measure of dose) versus immunodetection of the targeted gene product yields a dose-response curve for a given antisense compound.
An object of this invention is to develop antisense compounds targeted to the erbB-2 oncogene, which is amplified and overproduced in a large fraction of breast and other epithelial cancers.